1. Field of the Invention
The present invention relates to the production of liposomes containing lipophilic materials dissolved in the lipid layer of the liposome. More specifically, the invention is directed to the stabilization of oxidizable unsaturated lipophilic compounds by the encapsulation of the material in the lipidic layer of a liposome to retard or inhibit oxidation. The invention further relates to a lipophilic material stabilized by encapsulating the material in a liposome having a predetermined thermal transition temperature and shear resistance to withstand the physical treatments normally encountered in commercial food preparation. The liposomes can be used to introduce a readily oxidizable lipidic component to foods in a pure form. The liposomes offer a means whereby the lipophilic component can be easily dispersed in an aqueous phase. The liposomes can also be introduced to food compositions to slowly release the material dissolved in the lipidic layer over an extended period of time.
2. Brief Description of the Prior Art
Liposomes have been primarily used in recent years as delivery and carrier systems by encapsulating various compounds in the aqueous layer of the liposome. The emphasis of liposome development has been in the delivery of bioactive compounds. In the field of pharmaceutical and drug delivery, liposomes encapsulating drugs in the aqueous layer have been used successfully by suspending the liposome in a nonreactive carrier which can be injected directly into the treatment site to deliver an effective amount of the encapsulated drug.
Liposomes are essentially closed bilayer membranes in the form of vesicles or sacs containing an entrapped aqueous phase. Liposomes having a single bilayer membrane are referred to as unilamellar vesicles, whereas lipsomes having a number of concentric lipid bilayers separated by an aqueous phase are referred to as multilamellar vesicles. Recently, the attention on liposome preparation has focused on the liposomes with several bilayers, since these multilamellar vesicles demonstrate possibilities of encapsulating larger amounts of components than in the unilamellar vesicles.
Liposome preparation methods have been known for sometime. One of the earlier methods of preparing liposomes is described by Bangham et al. in J. Mol. Bio. Vol.13 pp. 238-252 (1965). According to that method, a phospholipid, such as phosphatidylcholine, is suspended in an organic solvent which is then evaporated to dryness, resulting in a waxy phospholipid deposit on the wall of the vessel. An aqueous phase containing a desired encapsulant is added to the reaction vessel, and mechanical means, such as ultrasonics, are used to disperse the resulting liposomes. The liposomes produced by this method are predominantly unilamellar vesicles (ULV). The liposomes have a lipidic bilayer, where the non-polar hydrophobic ends of the phospholipid orient themselves toward the center of the liposome bilayer, and the polar hydrophilic ends orient themselves towards the aqueous layer. Liposomes prepared according to this method are generally quite small, which restricts the ability to encapsulate large macromolecules.
As a consequence of these limitations, efforts have been made to increase the volume of the entrapped material. These methods have included the formation of inverse micelles or liposome precursors. The precursors are generally vesicles containing an aqueous phase surrounded by a monolayer of lipid molecules oriented so that the polar head groups are directed towards the aqueous phase. Liposome precursors are formed by adding the aqueous solution to be entrapped to a solution of an amphiphilic lipid in an organic solvent and sonicating the mixture. The organic phase is evaporated in the presence of the liposome precursors which associate with the excess lipid. The resulting liposomes are then dispersed in an aqueous phase. An example of such a process is disclosed in U.S. Pat. No. 4,224,179.
U.S. Pat. No. 4,235,871 to Papahadjopoulos discloses a reverse-phase evaporation process for making oligolamellar lipid vesicles, often referred to as reverse-phase evaporation vesicles or REVs. To prepare the REVs, a phospholipid is dissolved in an organic solvent contained in a flask, and the solvent is evaporated. The flask is purged with nitrogen, and the lipid is redissolved in the organic phase. The aqueous solution containing the material to be entrapped is added to the lipid solution. A homogeneous water-in-oil emulsion is then formed, and the organic solvent is evaporated until a gel remains. The gel is dispersed in an aqueous medium to convert the gel to a suspension. The preparation is finally dialyzed or centrifuged to remove non-encapsulated material and residual organic solvent.
In general, the larger a liposome, and the fewer layers it has, the more liquid it can encapsulate. Since the carrier liquid in which a liposome is dispersed can differ compositionally from that contained within the liposome itself, liposomes are potentially useful as biodegradable, relatively nontoxic vehicles for administering a component without risk of prematurely degrading the component, as might occur, for example, upon exposure to air or contact in the gastrointestinal tract.
Liposomes prepared from naturally occurring phospholipids have a tendency to be unstable during storage, thereby limiting their utility. This inherent instability results in a breakdown of the bilayers and the entrapped materials of the liposome diffusing into the surrounding liquid medium. This dispersing of the released materials can result in the contamination of the remaining liposomes by permeation of the materials from the surrounding medium into the liposome itself. The permeability characteristics of a liposome membrane bilayer can be altered by the addition of different stabilizers. For example, cholesterol can be mixed with other liposome-forming amphiphilic lipids to enhance the orientation of the amphiphilic lipids into a more orderly crystalline array. The more orderly arrangement stabilizes the bilayer and reduces permeation of materials.
A major use of liposomes to date has been in the medical field, by employing liposomes which entrap an effective amount of a water-soluble pharmaceutical compound and suspending the liposome in a pharmaceutically acceptable carrier. The advantages of using liposomes for this purpose arise from the fact that, when liposomes are injected into animals, they are taken up rapidly by the cells and intra-cellular lysosomes. Liposomes can be made to be relatively impermeable, and since they are rapidly taken up by the cells and removed from the circulatory system, the pharmaceutical remains concentrated in the liposome and unexposed to the plasma until such time as the liposomes are broken. This technology and knowledge primarily developed for the medical field offer alternative uses of liposomes, outside the delivery of pharmaceuticals, which have not been fully explored or utilized.
Liposomes may be prepared from a wide variety of lipidic compounds, including alkylamines, gangliosides, cardiolipin, and phospholipids Some of the more widely used lipids include the phospholipids wherein the phosphatidyl portion contains ester groups, for example C.sub.14 to C.sub.20, saturated or unsaturated, fatty acids. Specifically, liposomes may be prepared from any one or combination of dicetylphosphate, distearoylphosphatidic acid, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylinositol, mono and dialkyl esters of phosphatidylserine, and dioleoylphosphatidylglycerol. In addition, lysophosphatides, wherein the fatty acid ester of C.sub.1 or C.sub.2 has been removed, can be mixed with the above-noted lipids to form liposomes. Other synthetic phosphatidyl compounds used include those wherein a sulfonium, phosphonium, or quaternary ammonium polar head moiety has been modified by the addition of hydrocarbon groups, particularly alkyl groups.
In the preparation and processing of food products, it is desirable to include various components or additives such as enzymes, vitamins, preservatives, acidulants or edible oils. Some of these additives can be combined in foods only in limited amounts due to their unpleasant flavor characteristics. In addition, some desirable additives, such as some vegetable oils and fish oils, are readily oxidizable and develop an unpleasant odor and flavor after a relatively short period of time. Some additives simply degrade over time or after exposure to oxygen and light and become ineffective. As a result of these inherent adverse characteristics, efforts have been made to modify the additives in some manner to disguise the flavor or stabilize the components. These efforts have not employed the formation of food-compatible liposomes which contain readily oxidizable substances in the lipidic bilayer.
One example of the prior art liposomes is U.S. Pat. No. 4,508,703, which discloses that a partially or totally hydrophobic substance may be added into the lipidic bilayer of liposomes or hydrated lamellar phases. The hydrated lipidic lamellar phases are prepared from a pulverulent mixture for producing liposomes which are primarily intended to be used in the release and delivery of drugs, as well as in the cosmetic and food industries. A mixture of amphiphilic and hydrophobic lipid constituents is produced in an organic solvent and atomized to produce a dry pulverulent material. Suitable amphiphilic lipids include phospholipids, glycolipids or phosphoaminolipids, such as egg or soya lecithin, a phosphatidylserine, a cerebroside or a sphingomyelin. The hydrophobic or partially hydrophobic constituents include sterols or esters thereof, such as cholesterol, aliphatic fatty acids or esters, aliphatic fatty amines, fatty acid acylated amides, polypeptides, vitamins or extracts of animal or vegetable origin. These hydrophobic constituents are added to the amphiphilic lipid to improve the physical characteristics of the resulting liposome, such as wall strength, permeability and stability. The liposomes and hydrated lipidic lamellar phases are produced by dispersing the pulverulent mixture in a suitable aqueous medium. The aqueous medium may contain water-soluble substances, such as proteins for use in foods or sodium carboxylate pyrrolidone for use in cosmetics.
U.S. Pat. No. 4,222,891 discloses a method of producing microcapsules for use in pressure-sensitive and carbonless duplicating papers. The microcapsules are prepared from an aqueous solution of gelatin, an anionic colloid material and oil droplets dispersed in the solution, via coacervation. The oils employed are the conventional oils used in the carbonless paper industry, such as fish or lard oils, vegetable oils, mineral oils or the synthetic oils. There is no suggestion of using the encapsulated oils in food products or encapsulating to stabilize the oils and prevent premature oxidation.
U.S. Pat. No. 4,404,228 relates to a free-flowing, non-caking material prepared by spray drying or drum drying a lipid-containing suspension followed by a comminuting step. The lipidic constituents are contained in the interstitial spaces of water-insoluble porous protein particles. The lipids employed may include vegetable, animal or marine fats, such as coconut, palm, palm kernel, fish, soybean, sunflower or tallow oils. The resulting dry product is used primarily as a milk replacer. The lipids in this type of product are intended to be readily soluble in water. The lipidic constituents are not protected from oxidation and do not provide for a sustained release of the lipidic constituents over a period of time.
U.S. Pat. No. 4,528,226 discloses a stretchable sheet having a coating of rupturable microscopic capsules that release fragrances. Enclosed within the microcapsules are water-insoluble oils, such as olive oil, fish oils, vegetable oils, sperm oils or mineral oils.
"Accelerated ripening of cheese using liposome-encapsulated enzyme" Kirby et al. International Journal of Food Science and Technology Vol. 22, pp.355-75 (1987) discloses the use of liposomes, to encapsulate an enzyme in the aqueous phase, for accelerating the ripening of cheese. Encapsulating the enzyme within the liposomes stabilizes the enzyme, such that it remains active longer. This produces a 100-fold increase in rate over conventional methods of ripening cheese. The liposomes are prepared by the dehydration/rehydration vesicle procedure (DRV), using egg phosphatidylcholine and soya lecithin dissolved in chloroform:methanol. The solution is dried by rotary evaporation to form a thin film of the lipid around the inside of the flask. The lipid is dispersed by swirling with distilled water to produce an opalescent dispersion. Neutrase dissolved in distilled water is then added to the dispersion, and the dispersion is frozen by swirling in a liquid nitrogen bath. The frozen dispersion of liposomes is then freeze-dried. DRV's were formed by adding distilled water to give a complete suspension of the material.
"Encapsulation and stimulated release of enzymes using lecithin vesicles" Kirby et al. International Journal of Food Science and Technology, Vol. 22, pp. 707-723 (1987) discloses lecithin vesicles prepared by dehydration/rehydration to encapsulate enzymes such as lysozyme and pepsin. The stimulated release of the enzymes under varying conditions and release patterns were examined.
Fats and oils are known to undergo a flavor change during storage, particularly at elevated temperatures. The flavor change is also associated with the unpleasant odors which make the fats unsuitable for human consumption. This condition of fats and oils is generally referred to as rancidity. The fats and oils which are prone to rancidity generally contain significant quantities of fixed oils and other lipids which undergo oxidation leading to the formation of primary, secondary and tertiary oxidation products. These oxidation products are responsible for the unpleasant odors and flavors. Factors which affect the rate of oxidation include the amount of oxygen present, the degree of unsaturation, presence of antioxidants, presence of pro-oxidants such as copper and iron, presence of heme-containing molecules and lipoxidase, nature of packaging, U.V. light exposure, and temperature of storage.
Highly unsaturated fats and oils undergo autoxidation. Autoxidation consists generally of a free radical chain reaction having an induction period, rapid formation of hydroperoxides, and finally the formation of the highly odorous reaction products. In general terms, a free radical is formed by an initiator such as singlet oxygen, ozone or superoxide radicals formed from the interaction of ferrous contaminants with triplet oxygen. Other initiators include UV radiation and enzymes, such as xanthine oxidase, which produce superoxide radicals. Peroxides are then formed which, in turn, break down to form perhydroxyl radicals or hydrogen peroxide. Finally, the reaction proceeds to form nonreactive molecules. The reaction is further complicated, in that the fatty-acid peroxides can decompose into odorous volatile compounds, such as saturated and unsaturated aldehydes and ketones. These low molecular weight carbonyls are usually the cause of the unpleasant odor and flavor. These volatile oxidation products can sometimes be removed in the refining process during deodorization. The non-volatile products, such as fatty acid hydroperoxides, tend to result in a lower oxidative stability of the oils.
The degree of unsaturation and the types of unsaturated fatty acids present are also important considerations in the rate of oxidation. For example, for the 18 carbon atom fatty acids having degrees of unsaturation 18:0, 18:1, 18:2 and 18:3, the relative rates of oxidation are in the ratio of 1:100:1200:2500. Thus the fatty acid having three points of unsaturation is 2,500 times more reactive than the completely saturated fatty acid.
Fish oils, and in particular the omega-6 and omega-3 fatty acid fish oils, are examples of highly unsaturated oils which may contain as many as five points of unsaturation and are readily oxidized. The unstable nature of fish oils explains the reason for their poor odor and flavor characteristics after a relatively short period of storage time. Because the odor and flavor of fish oils are so strong, it is very difficult to remove the odorous components to produce a food product which does not taste and smell fishy. In addition, it is very difficult to produce a fish oil-containing product which has an extended shelf life. Fish oil contains a large amount of highly unsaturated fatty acids such as eicosatetraenoic acid, eicosapentaenoic acid and docosahexaenoic acid. These fatty acids have been shown to be beneficial in controlling the cholesterol level in blood and in preventing thrombotic disturbances.
Because omega-3 fish oils are believed to reduce the risk of heart disease in humans and to lower cholesterol levels in the blood by encouraging the body to excrete more cholesterol, efforts are being made to produce a fish oil product, without the prominent odor and taste of fish, which can be used as a substitute for the saturated fats contained in many foods. One such example is disclosed in U.S. Pat. No. 4,764,392 relating to a margarine composition having a high content of refined unsaturated fatty acids from fish oils. The refining step includes a three stage molecular distillation process under vacuum at elevated temperature. The margarine is prepared by combining the refined fish oil with the usual vegetable oils, water, whey solids and emulsifiers conventionally used in margarine manufacture. The prepared margarine preferably contains 5-15% by weight of the refined fish oil, but may contain up to 40%. Above 15% fish oil, the margarine develops a fish oil taste and smell. As with other refining processes, this process has the disadvantage of not removing all of the non-volatile components from the fish oil, leading to distinctive fish odor and taste. In addition, the fish oil is still prone to oxidation and will again develop the fish odor and taste, thereby limiting the shelf life of the product.
U.S. Pat. No. 4,564,475 discloses a stable composition comprising a fish oil and a phospholipid such as lecithin as a stabilizing agent.
Other methods to prevent oxidation of fish oils have included the use of antioxidants and the encapsulation of the oil in gelatin capsules. These methods have not been effective in stabilizing fish oils for extended periods of time, because gelatin capsules do not provide an oxygen-impermeable barrier. Gelatin capsules generally have a particle size of about 100.mu. and are too large to be readily dispersed in an aqueous suspension. Moreover, gelatin capsules of this size result in a grainy texture which is undesirable in a food product. These capsules are ruptured upon mastication, thereby releasing the fish oil into a food. The release of fish oil that is not stabilized imparts an undesirable fish odor and flavor to a food. The gelatin capsules are easily rupturable and thus cannot be mixed in a dispersion,.which is subjected to heat or high shear during processing, without release of the oil to the system.
In view of the favorable attributes of fish oil in the human diet, there is a need for a food-compatible product which is able to incorporate fish oil, but which is free from the distinctive fish odor or taste. There further is a need for a food-compatible composition which can stabilize labile components, prevent or inhibit oxidation of readily oxidizable lipophilic materials, and can be stored for extended periods of time without becoming rancid or losing its desirable characteristics.
The present invention is therefore directed to a food-compatible liposome composition containing highly oxidizable lipophilic materials dissolved in the lipid layer of a liposome. The liposomes prepared according to the invention provide a means to prevent or inhibit oxidation of the encapsulated lipophilic material. The invention is further directed to a suitable lipid-containing food product containing large amounts of the novel liposome composition but free from adverse effects on its flavor and odor.